All publications herein are incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. The following description includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.
The thyrotropin receptor (TSHR), a member of the G protein-coupled receptor (GPCR) superfamily, is a key regulator of thyroid function. Through the TSHR, the natural ligand, thyrotropin (TSH), and pathological autoantibodies (primarily stimulatory but occasionally blocking) modify cAMP generation by adenylyl cyclase and, consequently, many aspects of thyroid hormone synthesis and secretion (Vassart et al. 1992. The thyrotropin receptor and the regulation of thyrocyte function and growth. Endocr. Rev. 13:596-611; Rapoport et al. 1998. The thyrotropin receptor: Interaction with thyrotropin and autoantibodies. Endocr. Rev. 19:673-716.). TSH also induces thyrocyte growth and proliferation (Vassart et al. 1992. The thyrotropin receptor and the regulation of thyrocyte function and growth. Endocr. Rev. 13:596-611.). The TSHR is structurally similar to the receptors for the other anterior pituitary glycoprotein hormones (Vassart et al. 2004. A molecular dissection of the glycoprotein hormone receptors. Trends Biochem. Sci. 29:119-126), yet is functionally different in possessing relatively high constitutive activity in the absence of ligand (Van Sande et al. 1995. In Chinese hamster ovary K1 cells dog and human thyrotropin receptors activate both the cyclic AMP and the phosphatidylinositol 4,5-bisphosphate cascades in the presence of thyrotropin and the cyclic AMP cascade in its absence. Eur. J. Biochem. 229:338-343). This activity is partially constrained by the TSHR ectodomain (Zhang et al. 2000. The extracellular domain suppresses constitutive activity of the transmembrane domain of the human TSH receptor: implications for hormone-receptor interaction and antagonist design. Endocrinol. 141:3514-3517) that, therefore, functions as an inverse agonist (Vlaeminck-Guillem et al. 2002. Activation of the cAMP pathway by the TSH receptor involves switching of the ectodomain from a tethered inverse agonist to an agonist. Mol. Endocrinol. 16:736-746). Significant TSHR constitutive activity is a clinically relevant phenomenon in the treatment of thyroid carcinoma. After thyroidectomy, suppression of endogenous TSH secretion is a therapeutic goal to prevent or retard the proliferation or metastasis of residual thyroid carcinoma cells. However, even complete TSH suppression with supra-physiological doses of thyroxine cannot eliminate potentially harmful TSHR activity. Also, perhaps because of its inherent ‘noisiness’, the TSHR is highly susceptible to activation by a large variety of disease-inducing mutations, particularly within its serpentine region, as documented in a recent data base (Van Durme et al. 2006. GRIS: glycoproteinhormone receptor information system. Mol. Endocrinol. 20:2247-2255).
In recent years, understanding of TSHR structure and function has been greatly facilitated by the generation of murine (Loosfelt et al. 1992. Two-subunit structure of the human thyrotropin receptor. Proc. Natl. Acad. Sci. U.S.A. 89:3765-3769; Huang et al. 1993. The thyrotropin hormone receptor of Graves' disease: overexpression of the extracellular domain in insect cells using recombinant baculovirus, immunoaffinity purification and analysis of autoantibody binding. J. Mol. Endocrinol. 10:127-142; Johnstone et al. 1994. Monoclonal antibodies that recognize the native human thyrotropin receptor. Molec. Cell. Endocrinol. 105:R1-R9; Seetharamaiah et al. 1995. Generation and characterization of monoclonal antibodies to the human thyrotropin (TSH) receptor: antibodies can bind to discrete conformational or linear epitopes and block TSH binding. Endocrinol. 136:2817-2824; Costagliola et al. 1998. Genetic immunization against the human thyrotropin receptor causes thyroiditis and allows production of monoclonal antibodies recognizing the native receptor. J. Immunol. 160:1458-1465; Oda et al. 2000. Epitope analysis of the human thyrotropin (TSH) receptor using monoclonal antibodies. Thyroid 10:1051-1059; Sanders et al. 2002. Thyroid stimulating monoclonal antibodies. Thyroid 12:1043-1050; Costagliola et al. 2002. Generation of a mouse monoclonal TSH receptor antibody with stimulating activity. Biochem. Biophys. Res. Commun. 299:891-896; Gilbert et al. 2006. Monoclonal pathogenic antibodies to the TSH receptor in Graves' disease with potent thyroid stimulating activity but differential blocking activity activate multiple signaling pathways. J. Immunol. 176:5084-5092; Costagliola et al. 2004. Delineation of the discontinuous-conformational epitope of a monoclonal antibody displaying full in vitro and in vivo thyrotropin activity. Mol. Endocrinol. 18:3020-3024), hamster (Ando et al. 2002. A monoclonal thyroid-stimulating antibody. J. Clin. Invest 110:1667-1674) and human (Akamizu et al. 1999. Characterization of recombinant monoclonal antithyrotropin receptor antibodies (TSHRAbs) derived from lymphocytes of patients with Graves' disease: epitope and binding study of two stimulatory TSHRAbs. Endocrinol. 140:1594-1601; Sanders et al. S. B. 2003. Human monoclonal thyroid stimulating autoantibody. Lancet 362:126-128.) monoclonal antibodies (mAb). Of particular interest and importance are those mAb that are potent activators of the TSHR (Sanders et al. 2002. Thyroid stimulating monoclonal antibodies. Thyroid 12:1043-1050; Gilbert et al. 2006. Monoclonal pathogenic antibodies to the TSH receptor in Graves' disease with potent thyroid stimulating activity but differential blocking activity activate multiple signaling pathways. J. Immunol. 176:5084-5092; Costagliola et al. 2004. Delineation of the discontinuous-conformational epitope of a monoclonal antibody displaying full in vitro and in vivo thyrotropin activity. Mol Endocrinol. 18:3020-3024; Ando et al. 2002. A monoclonal thyroid-stimulating antibody. J. Clin. Invest 110:1667-1674; Sanders et al. S. B. 2003. Human monoclonal thyroid stimulating autoantibody. Lancet 362:126-128.) Monoclonal antibodies that function as competitive antagonists for thyroid stimulating autoantibodies (TSAb) have also received attention as possible therapeutic agents in Graves' disease (Lenzner et al. 2003. The effect of thyrotropin-receptor blocking antibodies on stimulating autoantibodies from patients with Graves' disease. Thyroid 13:1153-1161; Sanders et al. 2005. Characteristics of a monoclonal antibody to the thyrotropin receptor that acts as a powerful thyroid-stimulating autoantibody antagonist. Thyroid 15:672-682), although competition for TSH binding, a universal property of these blocking antibodies, will lead to hypothyroidism.
In recent years, the realization that many GPCR have ligand-independent constitutive activity to varying degrees has introduced a new classification of pharmacological agents. Besides agonists and antagonists, inverse agonists and neutral antagonists are now described. Inverse agonists reduce ligand-independent constitutive activity. Many classical competitive antagonists also have inverse agonist properties, unlike neutral antagonists (Bond et al. 2006. Recent developments in constitutive receptor activity and inverse agonism, and their potential for GPCR drug discovery. Trends Pharmacol. Sci. 27:92-96.). The search for inverse agonists as therapeutic agents to modulate GPCR expression is of much current interest (Ellis, C. 2004. The state of GPCR research in 2004. Nat. Rev. Drug Discov. 3:575, 577-575, 626.). The great majority of GPCR inverse agonists are small molecules, many used in clinical practice as drugs to reduce activity of receptors such as those for epinephrine, histamine, dopamine and angiotensin. In general, these agents bind to a pocket within the transmembrane helices. However, in a few cases, large antibody molecules have been generated that function as inverse agonists by binding to the extracellular loops of the β2-adrenergic (Peter et al. 2003. scFv single chain antibody variable fragment as inverse agonist of the beta2-adrenergic receptor. J. Biol. Chem. 278:36740-36747) and M2-muscarinic acetylcholine (Peter et al. 2004. Modulation of the M2 muscarinic acetylcholine receptor activity with monoclonal anti-M2 receptor antibody fragments. J. Biol. Chem. 279:55697-55706.) receptors.
Turning to the thyroid, the thyrotropin releasing hormone (TRH) receptors in the pituitary thyrotroph (Straub et al. 1990. Expression cloning of a cDNA encoding the mouse pituitary thyrotropin-releasing hormone receptor. Proc. Natl. Acad. Sci. U.S.A. 87:9514-9518; Itadani et at 1998. Cloning and characterization of a new subtype of thyrotropin-releasing hormone receptors. Biochem. Biophys. Res. Commun. 250:68-71; Cao et al. 1998. Cloning and characterization of a cDNA encoding a novel subtype of rat thyrotropin-releasing hormone receptor. J. Biol. Chem. 273:32281-32287.) and the TSHR in the thyrocyte (Nagayama at al. 1989. Molecular cloning, sequence and functional expression of the cDNA for the human thyrotropin receptor. Biochem. Biophys. Res. Comm. 165:1184-1190; Parmentier et al. 1989. Molecular cloning of the thyrotropin receptor. Science 246:1620-1622.) are both GPCRs. The former receptor, activated by a small ligand (TRH), has a small extracellular domain. The TSHR has a large ectodomain (397 amino acid residues after signal peptide deletion) consistent with its large (˜30 kDa), glycosylated ligand (TSH). Besides their natural ligands, small, synthetic molecules have been sought to modulate receptor function. For example, midozalam has been identified as an inverse agonist for the TRH receptor (Colson et al. 1998. A hydrophobic cluster between transmembrane helices 5 and 6 constrains the thyrotropin-releasing hormone receptor in an inactive conformation. Mol. Pharmacol. 54:968-978.) and another synthetic compound (org41821) is a partial agonist for the TSHR (Jaschke et al. 2006. A low molecular weight agonist signals by binding to the transmembrane domain of thyroid-stimulating hormone receptor (TSHR) and luteinizing hormone/chorionic gonadotropin receptor (LHCGR). J. Biol. Chem. 281:9841-9844.). Unlike TSH, org41821 interacts directly with TSHR transmembrane helices (Jaschke et al. 2006. A low molecular weight agonist signals by binding to the transmembrane domain of thyroid-stimulating hormone receptor (TSHR) and luteinizing hormone/chorionic gonadotropin receptor (LHCGR). J. Biol. Chem. 281:9841-9844.). A modification of this compound acts allosterically as an antagonist of TSH action (Moore et al. 2006. Evaluation of small-molecule modulators of the luteinizing hormone/choriogonadotropin and thyroid stimulating hormone receptors: structure-activity relationships and selective binding patterns. J. Med. Chem. 49:3888-3896.). However, no TSHR inverse agonist has been reported.
With the high constitutive activity of TSHR, the risks associated with suppressing TSH, and the side effect of other thyroid cancer and thyroid disease treatments, there exist a need in the art for an inverse agonist of TSHR, and a method to decrease the constitutive activity of TSHR.